Quinolinate-induced rat striatal excitotoxicity impairs endoplasmic reticulum Ca2+-ATPase function.

Excessive activation of NMDA glutamate receptors and the resulting loss of intracellular Ca(2+) homeostasis may be lethal (excitotoxic) to neurons. Such excitotoxicity can be induced in vivo by intrastriatal infusion of quinolinate, as this substance selectively activates NMDA receptors. The aim of the present research was to investigate whether the in vivo treatment of striatal tissue with quinolinate would lead to an early impairment of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity or mitochondrial Ca(2+) sequestration, two intracellular mechanisms involved in Ca(2+) homeostasis and signaling. Sodium quinolinate was infused intrastriatally into adult rats, and 6 h later the brains were removed and the corpora striata dissected. At this time point, striatal sections stained with Fluoro-Jade, a cellular marker of cell death, showed initial signs of neuronal degeneration. In addition, SERCA activity decreased 39% in relation to the activity observed in the control striata. A corresponding decrease of the same magnitude in (45)Ca(2+) uptake by striatal microsomes was also found in the treated striata. Western blot analysis did not indicate any decrease in SERCA levels in striatal tissue after quinolinate infusion. Mitochondrial Ca(2+) sequestration was still preserved in quinolinate-treated striatal tissue when the assay was carried out in the presence of physiological concentrations of ATP and Mg(2+). These results suggest that impairment of the SERCA function may be an early event in excitotoxicity.

3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.

Conformational changes of the nucleotide site of the plasma membrane Ca2+-ATPase probed by fluorescence quenching.

Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.